The function of the 7.5 kb plasmid, pCT, of Chlamydia trachomatis is still unknown. However the fact that this DNA element appears to be strongly conserved in C. trachomatis (both in terms of its presence in essentially all isolates and in terms of its is genetic structure) suggests that pCT may provide some, important, and perhaps advantageous, fiction to the chlamydial cell during its natural host infection. Recently, an open reading frame of pCT (ORF3; Comanducci et al., Plasmid 23:149–154, 1990) was expressed in E. coli as a recombinant fusion protein (Comanducci et al., J. Gen. Microbiology, 134:1083–1092, 1993). The expression system used added a 11-kDa N-terminal polypeptide of the MS2-bacteriophage polymerase to the 28-kDa polypeptide (pgp3) encoded by ORF3. The resulting 39-kDa product was used to show that pgp3 epitopes can be recognized on Western blots by antibodies present in sera from patients with chlamydial infections, but not in control sera from healthy donors. Following this observation, we subsequently tried to develop a serological test more suitable than the immunoblotting technique for obtaining reproducible and quantitative data from large numbers of clinical samples. We now report that an enzyme-linked immunoassay based on a new recombinant form of the pgp3 protein, can be used for assessing the prevalence of pgp3 antibodies in people with C. trachomatis infections.